A genomic clone encoding a portion of a novel, high-molecular-weight Dictyostelium myosin heavy chain has been obtained. Additional clones which span about half of the gene are being sequenced and clones spanning the rest of the gene are being sought. Efforts are underway to generate antibodies against this myosin and to create a corresponding null cell line. We continue to concentrate on the three "classic" Dictyostelium myosin I isoforms, myoB, myoC and myoD. Antibodies specific to each of these isoforms have been generated, and the proteins have been localized to a number of actin-rich cortical regions, such as the pseudopods at the leading edge of migrating cells. These studies should shed light on the possible functions of these myosin I isoforms in vivo. We have also mapped the nucleotide-insensitive actin binding site in the tail domain of myoC to the glycine- and proline-rich THy2 domain. We are attempting to define the functions of myoB, myoC and myoD in vivo using gene targeting/antisense (AS) techniques and behavioral analyses of mutant cell lines. Specifically, we have been analyzing single (myoB-), double (myoB-/myoD AS), and triple (myoB-/myoD AS/myoC AS) mutants of these classic myosin I isoforms. These mutants show a progressive impairment in chemotactic "streaming" behavior, which is due, at least in part, to slower rates of cell translocation (as determined by quantitative video microscopy). These mutants also show a progressive slowing in doubling times, and a corresponding suppression in both the rate of uptake of a fluid phase marker (~70% reduced in triple mutants) and efflux. Efforts are underway to identify where in the endocytic pathway myosin I functions. Finally, preliminary experiments suggest a progressive impairment in the process of phagocytosis in these mutants. MyoB has been chosen as a paradigm for structure/function analysis of the myosin I heavy chain. Altered forms of myoB are being introduced into myoB- null cells and tested as to function (rescue of phenotype) and localization. Cells expressing myoB which lacks the src-like THy3 domain are being analyzed and other mutants are in preparation. Acanthamoeba high-molecular-weight myosin I have been purified to homogeneity. The protein's Mg-ATPase is activated over 30 fold by F-actin (Km ~15 fM) and the protein translocates actin filaments at a rate of about0.1 micro m/sec. Rotary shadowed electron micrographs reveal a single head with a 50 nm long tail. Immunofluorescence localizations of HMWMI are in progress.